- Email: [email protected]
Augmented lipid accumulation in ethyl methyl sulphonate mutants of oleaginous microalga for biodiesel production
Accepted Manuscript Augmented lipid accumulation in ethyl methyl sulphonate mutants of oleaginous microalga for biodiesel production Juhi Mehtani, Neha Arora, Alok Patel, Priyanka Jain, Parul A. Pruthi, Kirshna Mohan Poluri, Vikas Pruthi PII: DOI: Reference:
S0960-8524(17)30389-9 http://dx.doi.org/10.1016/j.biortech.2017.03.108 BITE 17812
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
30 January 2017 15 March 2017 17 March 2017
Please cite this article as: Mehtani, J., Arora, N., Patel, A., Jain, P., Pruthi, P.A., Poluri, K.M., Pruthi, V., Augmented lipid accumulation in ethyl methyl sulphonate mutants of oleaginous microalga for biodiesel production, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.03.108
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Augmented lipid accumulation in ethyl methyl sulphonate mutants of oleaginous microalga for biodiesel production Juhi Mehtani1#, Neha Arora1#, Alok Patel1, Priyanka Jain2 Parul A. Pruthi1, Kirshna Mohan Poluri1 and Vikas Pruthi1* 1 2
Department of Biotechnology, Indian Institute of Technology Roorkee
Department of Chemical Engineering, Indian Institute of Technology Roorkee Roorkee, Uttarakhand-247667, India *Email id: [email protected]
Running Title: High lipid accumulating mutants of C. minutissima
*Corresponding Author:
Prof. Vikas Pruthi Email: [email protected]; [email protected] Phone: +91-1332-285530 Fax: 091-1332-273560
#
Authors Contributed Equally
1
Abstract The aim of this work was to generate high lipid accumulating mutants of Chlorella minutissima (CM) using ethyl methyl sulphonate (EMS) as a random chemical mutagen. Amid the 5% surviving cells after exposure to EMS (2M), three fast growing mutants (CM2, CM5, CM7) were selected and compared with wild type for lipid productivity and biochemical composition. Among these mutants, CM7 showed the maximum biomass (2.4 g/L) and lipid content (42 %) as compared to wild type (1.5 g/L; 27 %). Further, the mutant showed high photosynthetic pigments with low starch content signifying the re-allocation of carbon flux to lipid. The obtained mutant showed no visible morphological changes in comparison to its WT. The fatty acid profile showed increase in monounsaturated fatty acids while decreased saturated and polyunsaturated fatty acids signifying good quality biodiesel. The mutant strain thus obtained can be optimized further and applied for enhanced biodiesel production. Keywords: Chlorella minutissima, Ethyl Methyl Sulphonate, Lipid content, Mutant, Triacylglycerol
2
1. Introduction Energy crisis and global warming are the two major challenges confronting the sustainable development of the world. Development and deployment of renewable energy sources are therefore imperative to reduce the fossil fuel load and mitigate the greenhouse gases (GHG) emissions. Transportation sector contributes to the maximum fossil fuel usage and GHG emissions. In this regard, energy fuels derived from renewable energy biomass offer a sustainable solution. Bio-based fuels can be categorized into biodiesel (lipid derived) and bioethanol (sugar derived). Biodiesel has been recognized as an attractive source of diesel fuel and is gaining immense interest from researchers throughout the world. The main advantages of using biodiesel lies in terms of its non-toxic, biodegradable, negligible CO2 emission nature and its direct applicability in unmodified diesel engines (Lam & Lee 2012). Transesterification of triacylglycerols (TAGs) results in the formation of biodiesel or fatty acid methyl esters (FAMEs). These TAGs can be obtained from plants, waste cooking oil, fats or oleaginous microorganisms such as algae, yeast, fungi and bacteria. Amongst these, oleaginous microalgae have an edge over its counter parts due to its high growth rate, no seasonal/ climatic dependence, unique ability to grow on waste resources and no prerequisite for land (Singh et al., 2011). Microalgae (including cyanobacteria) can accumulate up to 60-70 % of lipids (TAGs) under adverse conditions such as nutritional limitation, temperature, salinity, UV etc. (Sharma et al., 2012). Nevertheless, increase in lipid, ceases the cell division leading to overall decrease in the lipid productivity (Arora et al., 2016a). Thus, to give microalgae derived biodiesel a commercial face, one needs to develop strategies to uncouple the lipid accumulation and algal growth. Genetic engineering and generation of high lipid accumulating mutants are two promising strategies to circumvent the low lipid productivity associated with microalgae. Genetic 3
modification of the algal genome by targeting the lipid and carbohydrate metabolism is a cumbersome process and is still in its infancy (Radakovits et al., 2012). Moreover, success rate of this method is subjective and varies from species to species, resulting in either stable or ephemeral transformation. On the other hand induction and selection of high lipid accumulating algal mutants is straightforward and well described in the literature (Sirikhachornkit et al., 2016). Mutagenesis can be achieved by treating the algal cells with different mutagenic agents like EMS (Ethyl Methyl Sulphonate), UV radiations, heavy ion irradiation, N-methyl-N′-nitro-Nnitrosoguanidine (Kamath et al., 2008; Ma et al., 2013; Beacham et al., 2015). Previously, researchers have generated random mutants of various microalgae and c yanobacteria such as Nannochloropsis sp, Chlamydomonas reinhardtti, Dunaliella tertiolecta, Chlorella sorokiniana, Chlorella pyrenoidosa, Synechocystis PCC 6803, Desmodesmus sp. to increase the lipid accumulation using the above mentioned mutagens/techniques. (Ma et al., 2013; Lee et al., 2014; Vonlanthen et al., 2015; Patel et al., 2016; Sachdeva et al., 2016; Sirikhachornkit et al., 2016; Zhang et al., 2016). Chlorella minutissima, an oleaginous green microalga has the knack to produce high quality biodiesel due to fast growth rate, uncomplicated farming and its ability towards producing higher lipid and biomass content (Tang et al., 2011). Further, Chlorella species have an additional advantage of not being easily contaminated by other strains of microalgae when cultivated in open ponds. Thus, to exploit C. minutissima, on an economic scale for the generation of biodiesel, amelioration in its growth and lipid content is necessary. Hence, in the present study, for the first time random EMS mutants of C. minutissima were generated and screened for high lipid productivity which was compared to wild strain. The first objective of the investigation was to optimize the concentration of EMS for generation of 4
fast growing and high lipid accumulating mutants of the respective microalga. Second, to correlate the high lipid content and the attenuation of other biochemical changes observed with respect to the wild type strain. Third, to analyze the quality of their accumulated lipids, estimation of biodiesel fatty acid profile, and fuel properties to ensure its applicability in diesel engines. 2. Materials and Methods Chlorella minutissima (MCC-27) was procured from the Center for the Conservation and Utilization of Blue Green Algae, Indian Agricultural Research Institute, New Delhi. All the chemicals used for the preparation of Bold’s Basal Media (BBM) were purchased from Himedia, India. The chemicals and solvents used in this study were of HPLC grade. The microalga was maintained in BBM as described earlier (Arora et al., 2016a). All the experiments were independently repeated three times (n=3) and the values are expressed as mean ± S.D. 2.1 Mutagenesis Ethyl methane sulfonate (EMS) was used to generate random high lipid accumulating mutants of C. minutissima. Briefly, 5 ml of wild type C. minutissima culture, pre cultivated for a period of 4 days in BBM was centrifuged at 8000 rpm for 10 min. The pellet obtained was washed with autoclaved BBM (3 times) and then suspended in 500 µL of 0.1M PBS (pH 7.0). To generate mutants, the cells were treated with six different EMS concentrations (0.45, 0.8, 1.4, 1.7, 2.0 and 2.4 M) and kept in the dark for 30 min at 27 °C. The activity of EMS was inactivated by treating the cell suspension with freshly prepared solution of 10 % (w/v) sodium thiosulphate and kept for 10 min in dark. These cells were then washed with 1mL of PBS (2 times), followed by resuspension in 1 ml BBM and incubated for 24 h in the dark at 27 °C. The survival rate of the 5
microbial cells was determined by visualizing and counting cells under a light microscope (60 X magnification) followed by serial dilution (10-5) and plating them on BBM agar plates to count the cells. The colonies that appeared after 2 weeks were counted and survival rate was calculated. The EMS concentration showing 5 % survival rate (2 M) was selected for generating mutants. These mutants were plated with appropriate dilutions on to BBM with 2 % agar and incubated under cool white fluorescent light (200 µmol m-2 s-1) at 27 °C for 7 days. The colonies that appeared after 3-4 days were then re-streaked on BBM plates till four generations to confirm the growth. These colonies were then grown in BBM for further analysis. 2.2 Growth and Dry cell weight (DCW) analysis Three fast growing mutants (CM2, CM5 and CM7) were able to survive the EMS treatment and therefore selected for growth/biochemical analysis. The mutants were cultivated in 500 mL Erlenmeyer flask containing 250 ml BBM for a period of 10 days under cool white fluorescent light (200 µmol m-2 s-1) at 27 °C. The growth rate was measured by taking the absorbance at 750 nm every 24 h and compared with the wild type. The dry cell weight (DCW; g/L) was measured on the 10th day by centrifuging the mutants and the wild type cells at 8000 rpm for 10 min. The wet biomass was dried at 80°C for 2 h in hot air oven and then gravimetrically weighed to get the DCW. Biomass productivity was then calculated using the following equation: Biomass Productivity = DCW/ cultivation time (days)
2.3 Starch and Lipid analysis
6
To estimate the starch content in the mutants as compared to the wild type, iodine staining was performed on the 10th day. Briefly, 500 µL of cell culture was mixed with 10 µL of iodine solution (10 mL distilled water, 0.5 g Iodine and 1g KI at 90 °C) and then centrifuged at 8000 rpm for 10 min (Takeshita et al., 2015). The cell pellet was washed thrice with 0.1% NaCl before visualizing under light microscope (EVOS-FL, Advance Microscopy Group, AMG, USA). Nile red staining was done to visualize lipid droplets inside the microalgal cells (Arora et al., 2016a). The total lipid was extracted using modified protocol of Bligh and Dyer (Bligh and Dyer, 1959; Arora et al., 2016a). Lipid productivity was then calculated according to the following equation: Lipid content (%) = Total lipid in grams / DCW Lipid Productivity = Lipid content (%) * Biomass Productivity/100 To detect the presence of triacylglycerols (TAGs) in the total lipids, thin layer chromatography (TLC) and Fourier Transform Infrared (FTIR) spectroscopy were performed. For TLC, 5 µl of the total lipid sample was spotted on the TLC plate (0.25-mm-thick silica gel G-60, F254 TLC plate; Merck, India) as described by Patel et al., 2015. FTIR spectra (Thermo Nicolet NEXUS, Maryland, USA) were collected over the wave number range 400–4000 cm-1 and triolein was used as standard (Miglio et al., 2013). 2.4 Biochemical and Morphological analysis In order to visualize the morphological changes in the mutants of C. minutissima as compared to its wild type, FESEM analysis was performed using previously described protocol (Arora et al., 2016b). The cell size (average of 100 cells) was also measured using ‘Image J 1.49a’ software and compared with the wild type (Patel et al., 2015).
7
The photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) were estimated on the 10th day by harvesting 2 ml of cell suspension, then adding 2 ml methanol (99.9 %) to the pellet and incubating it at 45 °C for 24 h. The supernatant was used to calculate the pigments as using the following equations: Chlorophyll a (Chl a; µg/ml) = 16.72 A665.2 – 9.16 A652.4 Chlorophyll b (Chl b; µg/ml) = 34.09 A652.4 – 15.28 A665.2 Carotenoids (µg/ml) = (1000 A470 –1.63 Chl a –104.9 Chl b)/221 where A is the absorbance (Lichtenthaler, 1987) Total carbohydrate content in the mutants and wild type microalgal cells were estimated using phenol sulfuric method (Dubois et al., 2002). CHNS elemental analyzer (Thermo-Scientific, USA) was used to estimate the total nitrogen content and then multiplied by a factor of 6.25 for total protein estimation. 2.5 Fatty acid methyl ester (FAME) analysis The total lipid extracted was transesterified using 6 % methanolic H2SO4 at 90 °C for 2 h (Arora et al., 2016a). The FAME phase was separated by mixing in hexane: water (1:2; v/v) followed by centrifugation at 8000 rpm for 5 min. The hexane containing FAME phase was expatriated out into fresh glass vial and 1µl of the FAME was used for Gas chromatography- mass spectroscopy analysis (GC-MS; Agilent Technologies, USA) with electron ionization (70 eV), DB-5 capillary column (30 mm *0.25 mm* 1 µm) using helium (1 ml/min) as carrier gas (Arora et al., 2016b). The biodiesel physical properties such as saponification value (mg KOH), Iodine value (g I2/100g), cetane number, degree of unsaturation (% wt), long chain saturation factor (% wt), high heating value (MJ/kg), kinematic viscosity (mm2 /s), density(g/cm3) and oxidative stability (h) 8
were estimated according to the empirical formulas and compared to plant oil methyl esters (Jatropha and Palm) as described previously (Arora et al., 2016a). Results and Discussion 3.1 Survival rate exhibited by EMS mutants Mutagenesis is a simple and robust technique to generate mutants of desirable characteristics such as high growth, lipid, carotenoid, thermo tolerance, heavy metal tolerance etc. (GarcíaVillada et al., 2004; Kamath et al., 2008; Ong et al., 2010; Ngampuak et al., 2016; Patel et al., 2016; Sirikhachornkit et al., 2016). High lipid accumulating microalgal strain development can lead to successful deployment of microalgal oil derived biodiesel on a large scale. EMS is a strong chemical mutagen as compared to UV radiations and has been widely used to generate microalgal mutants (Tillich et al., 2012). Keeping this in view, random mutants of C. minutissima were generated using EMS. The microalgal cells were treated with different concentrations of EMS (0.4 -2.4 M) to analyze the cell survival and optimize EMS concentration for 5 % survival rate (Supplementary Fig. 1). An apparent decline in the cells was observed when EMS concentration was periodically increased with 2.4 M showing 100 % cell lethality. EMS (2M) showed 5% survival rate which was then used as benchmark for further mutant generations. Amid the colonies that survived the 2 M dose of mutagenic agent, seven fast growing (CM1, CM2, CM3, CM4, CM5, CM6, CM7) colonies were selected and re-streaked to confirm the fast growth and mutation. Out of these seven fast growing mutants, only three colonies (CM2, CM5, and CM7) were able to survive on sub culturing (Supplementary Fig. 1). All these three rapid growing mutant strains were then evaluated for various growth/biochemical properties by cultivating them in 250 ml BBM separately.
9
3.2. Changes in the growth, dry cell weight and biomass productivity of the EMS mutants Based on the growth and appearance of microalgal colonies on BBM agar plates, all the three colonies (CM2, CM5 and CM7) were selected and compared to wild type for growth, DCW and productivity analysis. The growth rates of the mutants were recorded higher as compared to the wild type (Fig. 1). Among these mutants, CM7 showed the maximum absorbance at 750 nm followed by CM5 and CM2 as depicted in Fig. 1. The high growth rate is directly correlated with enhanced DCW and biomass productivity obtained by CM7 (2.4 ±0.2 g/L; 240 mg/L/d) followed by CM5 (2.2 ±0.25 g/L; 220 mg/L/d) and CM2 (1.67 ±0.2 g/L; 167 mg/L/d) respectively as compared to control (wild type species; 1.5 ±0.1 g/L; 150
mg/L/d) indicating that EMS
treatment has positively affected the growth metabolism (Table 1). The sharp increase in the growth rate of mutants may also indicate augmented metabolic rate in CM7. Indeed, similar sort of growth rate behavior has been reported for other green microalgae mutants such as Chlamydmonas reinhardtii, Synechocystis PCC 6803, C. pyrenoidosa NCIM 2738, Nannochloropsis sp.,
N. salina CCAP849/3 (Anandarajah et al., 2012; Lee et al., 2014;
Beacham et al., 2015; Patel et al., 2016; Sachdeva et al., 2016). In comparison to previous studies on EMS mutants of Nannochloropsis sp., Synechocystis PCC 6803 and Desmodesmus sp., CM7 showed higher biomass production (Table 1) (Doan and Obbard 2012; Patel et al., 2016; Zhang et al., 2016).
3.3 Changes in the starch and lipid composition in the EMS mutants The effects of EMS (2M) on starch and lipid content of CM2, CM5 and CM7 were observed by Iodine and Nile red staining. The mutants (CM5 and CM7) showed decreased starch granules 10
(blue spots) as compared to CM2 and control (Supplementary Fig. 2). Starch is a major form of carbohydrate storage and its synthesis directly competes with the lipid accumulation in microalgae as both pathways share common precursors (Sirikhachornkit et al., 2016; Patel et al., 2016). Their studies have shown that a decrease in the starch content or generation of starch less mutants led to increase in the lipid accumulation. Analogous to these studies, an increase in the intracellular lipid content in CM7 and CM5 was observed showing accumulation of lipid droplets (Supplementary Fig. 2). These qualitative results were in coherence with the total extracted lipid with CM7 showing ~1.5 folds enhanced lipid content (42 ± 0.25 %) and ~2.5 folds increase in the lipid productivity (101 ± 0.78 mg/L/d) followed by CM5 and CM2 as compared to the wild type (27 ± 0.2 % ; 41 mg/L/d) as shown in Table 1 and 2. A study on double mutants of Aradopsis thaliana suggested the possible role of ESV1 and LESV proteins involved in starch degradation (Feike et al., 2016). We envisage that EMS exposure resulted in activation of starch degrading enzymes, which in turn could result in generation of mutants with high lipid content. Furthermore, mutations at one or more locus in fatty acid biosynthesis genes could also delineate for higher lipid accumulation in mutants obtained. DGAT1 (Diacylglycerol acetyl transferase) enzyme plays a vital role in lipid synthesis as it catalyzes the conversion of diacylglycerol (DAG) to triacylglycerol (TAG) in the endoplasmic reticulum, and genetic change in such genes could hold a possible reason for increase in lipid content (Perry et al., 1999).
3.4 Biochemical and morphological variation in the EMS mutants To understand the alterations in the generated mutants at the biochemical level, the changes in the total carbohydrate, protein and photosynthetic pigments (chlorophyll a, chlorophyll b and
11
carotenoids) were estimated and compared with wild type (Table 2). Decrease in the starch content indicated that the level of carbohydrates in the mutants should be low as carbohydrate is majorly stored in the form of starch granules. Among the mutants, CM7 showed the lowest carbohydrate content (~1.7 folds lower) than wild type followed by CM5 and CM2 respectively (Table 2). The protein content in CM7 showed negligible change while CM2 and CM5 showed ~16-17 % decline as compared to wild type which deemed to be a possible explanation for increase in the growth rate of mutants (Table 2). Such dynamics of various biochemical properties could be the major reason that accounts for higher lipid accumulation in algal mutants. The mutants obtained on the plates showed considerable changes in the level of pigments present indicating the efficiency of photosynthesis receptors, which can be used to measure the physiological adaptivity. Chlorophyll content measures the physiological adaptivity of the mutants with the genetic variability supposed to be established due to the mutagenic agent (Fan et al., 2014). Interestingly, CM7 showed elevated levels of photosynthetic pigments such as chlorophyll a (~1.3 folds), chlorophyll b (~1.2 folds) as compared to wild type while similar carotenoids content were observed in them (Table 2). This was also evident from the color of the mutants which appeared more dark green as compared to wild type (Supplementary Fig 1). Similar results were obtained in EMS treated mutants of Synechocystis PCC 6803 which reported increase in the pigments of the mutants (Patel et al., 2016). This increase in biomass as well as chlorophyll content in CM7 could account for multifarious changes in the genome of the algae strain which may result in such phenotypic variations. Further, an increase in the level of chlorophyll a could result in the up regulation in the activity of ACCase (committing step for fatty acid synthesis) which could be a possible explanation for high lipid content (Lv et al., 2010). Presence of LHCII complex marks a remarkable effect on the photosynthetic ability of 12
microalgae (Drop et al., 2014). The data suggested that either due to modification in starch synthesis enzymes or polygenic point mutation in lipid generating pathway could be a possible reason for high lipid accumulating mutants. Such diverse phenotypic effects of EMS could also attribute for the polygenic mutation caused in C. minutissima. Microscopic imaging (light microscopy and FESEM) were done to analyze the alterations in the appearance and cell size of the mutants (Supplementary Fig. 3). Images visualized showed no change in the appearance of the mutants cells (CM2, CM5 and CM7) as compared to the wild type. It could be possible that EMS led to point mutation in the algal cells resulting in changes at the molecular level with no visible cell size and morphological changes (Table 2). 3.5 Analysis of triacylglycerol in the total lipid extracted and FAME profile of EMS mutant Transesterification of TAGs results in the formation of biodiesel. For high grade biodiesel quality, TAG content present in microalgae cell plays the key role (Krohn et al., 2011). Thus, the total lipid accumulated in mutant and wild type and the highest lipid content mutant CM7 were analyzed for TAG quality and properties using various biophysical techniques. Visible brown spot was observed on TLC plate which confirmed the presence of TAG in CM7 and wild type using Triolein as a control (Supplementary Fig. 4). Further, FTIR spectrum showed distinctive peaks at C-H bond vibrations mainly aliphatic chains of CH3 and CH2 at ~2925-2850 cm-1. Peak at ~1745 cm-1 , ~ 1160 cm-1 confirmed the presence of C=O and C-O-C stretch which correspond to the ester groups of TAGs. Absence of bands in the spectral region 4000–3450 cm-1 indicated lack of hydroxyl and amine groups (Supplementary Fig. 4). Results obtained here were in coherence with the study of Miglio et al. indicating presence of TAGs in the total lipid extracted (Miglio et al., 2013).
13
The FAME profile of transesterified samples from CM7 and wild type revealed the presence of myristic acid (C14:0), palmitic acid (C16:0), hexadecadienoic acid methyl ester (C16:1), heptadecanoic acid (C17:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and eicosanoic acid (C20:0) as shown in Fig. 2. The mutant strain (CM7) as compared to wild type showed decrease in saturated fatty acids (~1.1 folds), polyunsaturated fatty acids (~1.75 folds) while increase in mono-saturated fatty acids particularly C18:1 by ~1.6 folds respectively. This decreased level of poly unsaturated fatty acids (PUFA) can increase the storage life of the derived biodiesel from the mutant CM7, as high levels of the same (PUFA) could lead to oxidation resulting in the formation of epoxy fatty acids (Patel et al., 2016). Elevated content of mono unsaturated fatty acids (MUFA) recorded in CM7 could be responsible for ameliorating the use of biodiesel at low temperatures on account of its liquidity as has been reported by Zhang et al., 2016 while working on EMS mutants of Desmodesmus sp. Therefore, increased MUFA along with decreased PUFA content elucidate for better quality of biodiesel in CM7 as compared to wild type. The biodiesel physical properties obtained of the mutant (CM7) were better than wild type (WT) and abided by the standards given by ASTM D6751-02 and EN 14214 respectively (Table 3). CM7 mutant showed a low Iodine value (60 g I2/100g) with a high cetane number (67) and high heating value (41 MJ/kg) and oxidative stability (11 h) indicating shorter ignition delay time, better cold start behavior, smooth engine run, long shelf life and complete combustion leading to reduced gaseous and particulate emissions as compared to WT (Ramos et al., 2009; Arora et al., 2016a). Further, the density and kinematic viscosity of both WT and CM7 were within the permissible range (3.5 to 5.0 mm2/s; 0.86 to 0.90 g/cm3). The obtained biodiesel physical
14
properties were also comparable to plant oil methyl esters (Jatropha and Palm) as shown in Table 3. 4. Conclusion This study reported the generation of mutant strain (CM7) with higher growth rate and elevated level of lipid content of 42 ± 0.2 % with no significant changes in its morphology and size. The mutant exhibited reduced starch (carbohydrate content) and increased photosynthetic pigments that can be attributed to its high growth rate. However, to deduce the molecular level changes related to the enhancement of lipid product in the CM7 mutant strain, studies comprising of metabolomics and proteomic are quintessential. Further, in order to explore the mutant’s field applicability, environmental and ecological acceptability must be established. Acknowledgement Authors are thankful for financial support by the Department of Biotechnology, Govt. of India, BioCare Programme, DBT Sanction No. 102/IFD/SAN/3539/2011-2012 (Grant No. DBT-608BIO) to PAP and DBT-SRF to NA (Grant No. 7001-35-44). KMP acknowledges the receipt of DBT-IYBA fellowship and SERB-LS young scientist award. Figure Legends: Fig. 1: Growth curves of C. minutissima wild type (WT) and its mutants (CM2, CM5 and CM7) grown in BBM for 10 days. Fig 2: FAME profile of the extracted lipid from the wild type and CM7.
References
15
1. Anandarajah, K., Mahendraperumal, G., Sommerfeld, M., Hu, Q., 2012. Characterization of microalga Nannochloropsis sp. mutants for improved production of biofuels. Appl. Energy 96, 371–377. doi:10.1016/j.apenergy.2012.02.057 2. Arora, N., Patel, A., Pruthi, P.A., Pruthi, V., 2016a. Synergistic dynamics of nitrogen and phosphorous influences lipid productivity in Chlorella minutissima for biodiesel production. Bioresour. Technol. 213, 79-87. 3. Arora, N., Patel, A., Pruthi, P.A., Pruthi, V., 2016b. Boosting TAG accumulation with improved biodiesel production from novel oleaginous microalgae Scenedesmus sp. IITRIND2 utilizing waste sugarcane bagasse aqueous extract (SBAE). Appl Biochem Biotechnol. 180,422 109-121. 4. Beacham, T.A., Macia, V.M., Rooks, P., White, D.A., Ali, S.T., 2015. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis. Biotechnol. Reports 7, 87–94. doi:10.1016/j.btre.2015.05.007 5. Bligh, E., Dyer, W., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. 37. 6. Doan, T.T.Y., Obbard, J.P., 2012. Enhanced intracellular lipid in Nannochloropsis sp. via random mutagenesis and flow cytometric cell sorting. Algal Res. 1, 17–21. doi:10.1016/j.algal.2012.03.001 7. Drop, B., Webber-Birungi, M., Yadav, S.K.N., Filipowicz-Szymanska, A., Fusetti, F., Boekema, E.J., Croce, R., 2014. Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii. Biochim. Biophys. Acta Bioenerg. 1837, 63–72. doi:10.1016/j.bbabio.2013.07.012 8. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 2002. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. doi:10.1021/ac60111a017 9. Fan, J., Cui, Y., Wan, M., Wang, W., Li, Y., 2014. Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors. Biotechnol Biofuels 7, 17. doi:10.1186/1754-6834-7-17 10. Feike, D., Seung, D., Graf, A., Bischof, S., Ellick, T., Coiro, M., Soyk, S., Eicke, S., Mettler-Altmann, T., Lu, K.-J., Trick, M., Zeeman, S.C., Smith, A.M., 2016. The starch granule-associated protein EARLY STARVATION1 is required for the control of starch 16
degradation
in
Arabidopsis
thaliana
leaves.
Plant
Cell
28,
1472–1489.
doi:10.1105/tpc.16.00011 11. García-Villada, L., Rico, M., Altamirano, M., Sánchez-Martín, L., López-Rodas, V., Costas, E., 2004. Occurrence of copper resistant mutants in the toxic cyanobacteria Microcystis aeruginosa: Characterization and future implications in the use of copper sulphate as algaecide. Water Res. 38, 2207–2213. doi:10.1016/j.watres.2004.01.036 12. Krohn, B.J., McNeff, C. V., Yan, B., Nowlan, D., 2011. Production of algae-based biodiesel using the continuous catalytic Mcgyan process. Bioresour. Technol. 102, 94– 100. doi:10.1016/j.biortech.2010.05.035 13. Lam, M.K., Lee, K.T., 2012. Microalgae biofuels: A critical review of issues, problems and
the
way
forward.
Biotechnol.
Adv.
30,
673–690.
doi:10.1016/j.biotechadv.2011.11.008 14. Lee, B., Choi, G.G., Choi, Y.E., Sung, M., Park, M.S., Yang, J.W., 2014. Enhancement of lipid productivity by ethyl methane sulfonate-mediated random mutagenesis and proteomic analysis in Chlamydomonas reinhardtii. Korean J. Chem. Eng. 31, 1036–1042. doi:10.1007/s11814-014-0007-5 15. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. doi:10.1016/0076-6879(87)48036-1 16. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. doi:10.1016/j.biortech.2010.03.120 17. Ma, Y., Wang, Z., Zhu, M., Yu, C., Cao, Y., Zhang, D., Zhou, G., 2013. Increased lipid productivity and TAG content in Nannochloropsis by heavy-ion irradiation mutagenesis. Bioresour. Technol. 136, 360–367. doi:10.1016/j.biortech.2013.03.020 18. Miglio, R., Palmery, S., Salvalaggio, M., Carnelli, L., Capuano, F., Borrelli, R., 2013. Microalgae triacylglycerols content by FT-IR spectroscopy. J. Appl. Phycol. 25, 1621– 1631. doi:10.1007/s10811-013-0007-6 19. Ngampuak, V., Chookaew, Y., Dejtisakdi, W., 2016. KU11 Mutants affected for growth rate , cell accumulation and biomass. WASET. 10, 420–424. 20. Ong, S.C., Kao, C.Y., Chiu, S.Y., Tsai, M.T., Lin, C.S., 2010. Characterization of the thermal-tolerant mutants of Chlorella sp. with high growth rate and application in 17
outdoor
photobioreactor
cultivation.
Bioresour.
Technol.
101,
2880–2883.
doi:10.1016/j.biortech.2009.10.007 21. Patel, A., Sindhu, D.K., Arora, N., Singh, R.P., Pruthi, V., Pruthi, P.A., 2015. Biodiesel production from non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp using oleaginous yeast Rhodosporidium kratochvilovae HIMPA1. Bioresour. Technol. 197, 91– 98. doi:10.1016/j.biortech.2015.08.039 22. Patel, V.K., Maji, D., Pandey, S.S., Rout, P.K., Sundaram, S., Kalra, A., 2016. Rapid budding EMS mutants of Synechocystis PCC 6803 producing carbohydrate or lipid enriched biomass. Algal Res. 16, 36–45. doi:10.1016/j.algal.2016.02.029 23. Perry, H.J., Bligny, R., Gout, E., Harwood, J.L., 1999. Changes in Kennedy pathway intermediates associated with increased triacylglycerol synthesis in oil-seed rape. Phytochemistry 52, 799–804. doi:10.1016/S0031-9422(99)00294-0 24. Radakovits, R., Jinkerson, R.E., Fuerstenberg, S.I., Tae, H., Settlage, R.E., Boore, J.L., Posewitz, M.C., 2012. Draft genome sequence and genetic transformation of the oleaginous
alga
Nannochloropis
gaditana.
Nat.
Commun.
3,
686.
doi:10.1038/ncomms1688 25. Ramos, M.J, Fernández, C.M., Casas, Rodríguez L., Pérez, Á., 2009. Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour. Technol, 100, 261– 268. doi.org/10.1016/j.biortech.2008.06.039 26. Sachdeva, N., Gupta, R.P., Mathur, A.S., Tuli, D.K., 2016. Enhanced lipid production in thermo-tolerant mutants of Chlorella pyrenoidosa NCIM 2738. Bioresour. Technol. 221, 576–587. doi:10.1016/j.biortech.2016.09.049 27. Sandesh Kamath, B., Vidhyavathi, R., Sarada, R., Ravishankar, G.A., 2008. Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus
pluvialis
mutants.
Bioresour.
Technol.
99,
8667–8673.
doi:10.1016/j.biortech.2008.04.013 28. Sharma, P., Jha, A.B., Dubey, R.S., Pessarakli, M., 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot. 2012, 1–26. doi:10.1155/2012/217037 29. Singh, A., Olsen, S.I., Nigam, P.S., 2011. A viable technology to generate thirdgeneration biofuel. J. Chem. Technol. Biotechnol. 86, 1349–1353. doi:10.1002/jctb.2666 18
30. Sirikhachornkit, A., Vuttipongchaikij, S., Suttangkakul, A., Yokthongwattana, K., Juntawong, P., Pokethitiyook, P., Kangvansaichol, K., Meetam, M., 2016. Increasing the triacylglycerol content in Dunaliella tertiolecta through isolation of starch-deficient mutants. J. Microbiol. Biotechnol. 26, 854–866. doi:10.4014/jmb.1510.10022 31. Takeshita, T., Takeda, K., Ota, S., Yamazaki, T., Kawano, S., 2015. A simple method for measuring the starch and lipid contents in the cell of microalgae. Cytologia (Tokyo). 80, 475–481. doi:10.1508/cytologia.80.475 32. Tang, H., Chen, M., Garcia, M.E.D., Abunasser, N., Ng, K.Y.S., Salley, S.O., 2011. Culture of microalgae Chlorella minutissima for biodiesel feedstock production. Biotechnol. Bioeng. 108, 2280–2287. doi:10.1002/bit.23160 33. Tillich, U.M., Lehmann, S., Schulze, K., Dühring, U., Frohme, M., 2012. The Optimal Mutagen Dosage to Induce Point-Mutations in Synechocystis sp. PCC6803 and Its Application
to
Promote
Temperature
Tolerance.
PLoS
One
7,
1–8.
doi:10.1371/journal.pone.0049467 34. Vonlanthen, S., Dauvillée, D., Purton, S., 2015. Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids. Algal Res. 12, 109– 118. doi:10.1016/j.algal.2015.08.008 35. Zhang, Y., He, M., Zou, S., Fei, C., Yan, Y., Zheng, S., Rajper, A.A., Wang, C., 2016. Breeding of high biomass and lipid producing Desmodesmus sp. by Ethylmethane sulfonate-induced
mutation.
Bioresour.
doi:10.1016/j.biortech.2016.01.120
19
Technol.
207,
268–275.
Fig. 1
20
21
Table 1: Comparison of previously reported algal EMS mutants with the current study
Microalgae
WT/ Mutant
Biomass (g/L)
Lipid (%)
Nannochloropsis sp.
WT Mutant WT Mutant WT Mutant WT Mutant WT Mutant WT Mutant (CM2) Mutant (CM5) Mutant (CM7)
0.20 ± 0.02 0.22 ± 0.08 2.7 3.1 2.9 ± 0.002 2.46 ± 0.006 0.534 ± 0.014 0.778 ± 0.012 1.182 ± 0.010 1.397 ± 0.011 1.5±0.1 1.67±0.2
Chlorella pyrenoidosa Desmodesmus sp. Synechocystis PCC 6803 C. minutissima
34 ± 3.8 50.8 ± 6.8 47.4 ± 0.02 48.3 ± 0.02 21.78 ± 0.023 39.89 ± 0.004 40.41 48.41 12.97 32.80 27±0.5 32±0.25
Biomass Productivity (mg/L/d) 11.12 12.22 456 ± 0.01 517 ± 0.03 580 492 28.15 ± 0.75 40.95 ± 0.64 84.43 99.78 150±1.2 167±2.3
Lipid Productivity (mg/L/d) 3.77 6.20 213 249.55 105.27 196.26 495 778.10 10.95 32.72 40.5±1.4 53.44±2.5
2.2±0.25
36±0.3
220±1.3
79.2±2.4
2.4±0.2
42±0.12
240±2.1
100.8±1.5
22
R
Table 2: Relative changes in cell size (µm), total biochemical composition (lipid, carbohydrate and protein) and photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) in C. minutissima and its mutants (CM2, CM5 and CM7). WT/Mutant
Wild type CM2 CM5 CM7
Cell size (µm) 5.1±0.1 4.8±0.3 4.9±0.2 5.0±0.3
Biochemical composition Total lipid (%) 27± 0.5 32±0.3 36±0.3 42±0.1
Total carbohydrate (%) 30.5±1.3 29.2±0.4 28.9±0.3 17.5±0.3
Photosynthetic pigments Total protein (%) 42.5±2.1 38.8±1.3 35.1±1.5 40.5±2.3
Chlorophyll a (µg/ml) 6.3±0.1 6.4±0.2 5.4±0.1 8.1±0.2
Chlorophyll b (µg/ml) 4.2±0.2 4.3±0.1 3.7±0.2 5.1±0.1
Carotenoids (µg/ml) 0.34±0.01 0.43±0.02 0.31±0.01 0.36±0.02
Table 3: Summary of the biodiesel physical properties of C. minutissima (Wild type and Mutant CM7) obtained in the current study and compared to ASTM D6751-52, EN 14214 biodiesel standards and plant oil methyl esters. Standard fuel parameters Physical properties Saponification value (mg KOH) Iodine value (g I2/100g) Cetane number Degree of unsaturation (% wt) Long chain saturation factor (% wt) High heating value (MJ/kg) Kinematic viscosity (mm2/s) Density(g/cm3 ) Oxidative stability (h)
ASTM D6751-02 47 min 1.9 to 6.0 -
C. minutissima
EN 14214 120 (max) 51 (min) 3.5 to 5.0 0.86 to 0.90 ≥6
23
Wild type 184±2 70±4 58±3 82±2 12±2 40±1 4.4±0.2 0.87±0.01 7±0.2
CM7 (Mutant) 184±3 60±2 67±1 75±2 10±0.4 41±0.5 4.3±0.1 0.87±0.02 11±0.1
Plant oil methyl esters JME 96 57 4.3 0.88 4
PME 49 42 4.5 0.92 16
-No standard limit designated by ASTM D6751-02 and EN 14214 biodiesel standards, JME= Jatropha methyl ester, PME= Palm oil methyl ester **As the values were calculated theoretically, the values were rounded off to whole numbers
24
Highlights • • • • •
High lipid accumulating mutants of C. minutissima using ethyl methyl sulphonate. Mutants exhibited enhanced growth rate and photosynthetic pigments Reduced starch content indicated re-allocation of carbon flux to lipids. Increased MUFA and decreased SFA/ PUFA content in mutant CM7. High MUFA improves shelf life, cold flow properties and cetane number of biodiesel.
25
Graphical Abstract
26
S0960-8524(17)30389-9 http://dx.doi.org/10.1016/j.biortech.2017.03.108 BITE 17812
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
30 January 2017 15 March 2017 17 March 2017
Please cite this article as: Mehtani, J., Arora, N., Patel, A., Jain, P., Pruthi, P.A., Poluri, K.M., Pruthi, V., Augmented lipid accumulation in ethyl methyl sulphonate mutants of oleaginous microalga for biodiesel production, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.03.108
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Augmented lipid accumulation in ethyl methyl sulphonate mutants of oleaginous microalga for biodiesel production Juhi Mehtani1#, Neha Arora1#, Alok Patel1, Priyanka Jain2 Parul A. Pruthi1, Kirshna Mohan Poluri1 and Vikas Pruthi1* 1 2
Department of Biotechnology, Indian Institute of Technology Roorkee
Department of Chemical Engineering, Indian Institute of Technology Roorkee Roorkee, Uttarakhand-247667, India *Email id: [email protected]
Running Title: High lipid accumulating mutants of C. minutissima
*Corresponding Author:
Prof. Vikas Pruthi Email: [email protected]; [email protected] Phone: +91-1332-285530 Fax: 091-1332-273560
#
Authors Contributed Equally
1
Abstract The aim of this work was to generate high lipid accumulating mutants of Chlorella minutissima (CM) using ethyl methyl sulphonate (EMS) as a random chemical mutagen. Amid the 5% surviving cells after exposure to EMS (2M), three fast growing mutants (CM2, CM5, CM7) were selected and compared with wild type for lipid productivity and biochemical composition. Among these mutants, CM7 showed the maximum biomass (2.4 g/L) and lipid content (42 %) as compared to wild type (1.5 g/L; 27 %). Further, the mutant showed high photosynthetic pigments with low starch content signifying the re-allocation of carbon flux to lipid. The obtained mutant showed no visible morphological changes in comparison to its WT. The fatty acid profile showed increase in monounsaturated fatty acids while decreased saturated and polyunsaturated fatty acids signifying good quality biodiesel. The mutant strain thus obtained can be optimized further and applied for enhanced biodiesel production. Keywords: Chlorella minutissima, Ethyl Methyl Sulphonate, Lipid content, Mutant, Triacylglycerol
2
1. Introduction Energy crisis and global warming are the two major challenges confronting the sustainable development of the world. Development and deployment of renewable energy sources are therefore imperative to reduce the fossil fuel load and mitigate the greenhouse gases (GHG) emissions. Transportation sector contributes to the maximum fossil fuel usage and GHG emissions. In this regard, energy fuels derived from renewable energy biomass offer a sustainable solution. Bio-based fuels can be categorized into biodiesel (lipid derived) and bioethanol (sugar derived). Biodiesel has been recognized as an attractive source of diesel fuel and is gaining immense interest from researchers throughout the world. The main advantages of using biodiesel lies in terms of its non-toxic, biodegradable, negligible CO2 emission nature and its direct applicability in unmodified diesel engines (Lam & Lee 2012). Transesterification of triacylglycerols (TAGs) results in the formation of biodiesel or fatty acid methyl esters (FAMEs). These TAGs can be obtained from plants, waste cooking oil, fats or oleaginous microorganisms such as algae, yeast, fungi and bacteria. Amongst these, oleaginous microalgae have an edge over its counter parts due to its high growth rate, no seasonal/ climatic dependence, unique ability to grow on waste resources and no prerequisite for land (Singh et al., 2011). Microalgae (including cyanobacteria) can accumulate up to 60-70 % of lipids (TAGs) under adverse conditions such as nutritional limitation, temperature, salinity, UV etc. (Sharma et al., 2012). Nevertheless, increase in lipid, ceases the cell division leading to overall decrease in the lipid productivity (Arora et al., 2016a). Thus, to give microalgae derived biodiesel a commercial face, one needs to develop strategies to uncouple the lipid accumulation and algal growth. Genetic engineering and generation of high lipid accumulating mutants are two promising strategies to circumvent the low lipid productivity associated with microalgae. Genetic 3
modification of the algal genome by targeting the lipid and carbohydrate metabolism is a cumbersome process and is still in its infancy (Radakovits et al., 2012). Moreover, success rate of this method is subjective and varies from species to species, resulting in either stable or ephemeral transformation. On the other hand induction and selection of high lipid accumulating algal mutants is straightforward and well described in the literature (Sirikhachornkit et al., 2016). Mutagenesis can be achieved by treating the algal cells with different mutagenic agents like EMS (Ethyl Methyl Sulphonate), UV radiations, heavy ion irradiation, N-methyl-N′-nitro-Nnitrosoguanidine (Kamath et al., 2008; Ma et al., 2013; Beacham et al., 2015). Previously, researchers have generated random mutants of various microalgae and c yanobacteria such as Nannochloropsis sp, Chlamydomonas reinhardtti, Dunaliella tertiolecta, Chlorella sorokiniana, Chlorella pyrenoidosa, Synechocystis PCC 6803, Desmodesmus sp. to increase the lipid accumulation using the above mentioned mutagens/techniques. (Ma et al., 2013; Lee et al., 2014; Vonlanthen et al., 2015; Patel et al., 2016; Sachdeva et al., 2016; Sirikhachornkit et al., 2016; Zhang et al., 2016). Chlorella minutissima, an oleaginous green microalga has the knack to produce high quality biodiesel due to fast growth rate, uncomplicated farming and its ability towards producing higher lipid and biomass content (Tang et al., 2011). Further, Chlorella species have an additional advantage of not being easily contaminated by other strains of microalgae when cultivated in open ponds. Thus, to exploit C. minutissima, on an economic scale for the generation of biodiesel, amelioration in its growth and lipid content is necessary. Hence, in the present study, for the first time random EMS mutants of C. minutissima were generated and screened for high lipid productivity which was compared to wild strain. The first objective of the investigation was to optimize the concentration of EMS for generation of 4
fast growing and high lipid accumulating mutants of the respective microalga. Second, to correlate the high lipid content and the attenuation of other biochemical changes observed with respect to the wild type strain. Third, to analyze the quality of their accumulated lipids, estimation of biodiesel fatty acid profile, and fuel properties to ensure its applicability in diesel engines. 2. Materials and Methods Chlorella minutissima (MCC-27) was procured from the Center for the Conservation and Utilization of Blue Green Algae, Indian Agricultural Research Institute, New Delhi. All the chemicals used for the preparation of Bold’s Basal Media (BBM) were purchased from Himedia, India. The chemicals and solvents used in this study were of HPLC grade. The microalga was maintained in BBM as described earlier (Arora et al., 2016a). All the experiments were independently repeated three times (n=3) and the values are expressed as mean ± S.D. 2.1 Mutagenesis Ethyl methane sulfonate (EMS) was used to generate random high lipid accumulating mutants of C. minutissima. Briefly, 5 ml of wild type C. minutissima culture, pre cultivated for a period of 4 days in BBM was centrifuged at 8000 rpm for 10 min. The pellet obtained was washed with autoclaved BBM (3 times) and then suspended in 500 µL of 0.1M PBS (pH 7.0). To generate mutants, the cells were treated with six different EMS concentrations (0.45, 0.8, 1.4, 1.7, 2.0 and 2.4 M) and kept in the dark for 30 min at 27 °C. The activity of EMS was inactivated by treating the cell suspension with freshly prepared solution of 10 % (w/v) sodium thiosulphate and kept for 10 min in dark. These cells were then washed with 1mL of PBS (2 times), followed by resuspension in 1 ml BBM and incubated for 24 h in the dark at 27 °C. The survival rate of the 5
microbial cells was determined by visualizing and counting cells under a light microscope (60 X magnification) followed by serial dilution (10-5) and plating them on BBM agar plates to count the cells. The colonies that appeared after 2 weeks were counted and survival rate was calculated. The EMS concentration showing 5 % survival rate (2 M) was selected for generating mutants. These mutants were plated with appropriate dilutions on to BBM with 2 % agar and incubated under cool white fluorescent light (200 µmol m-2 s-1) at 27 °C for 7 days. The colonies that appeared after 3-4 days were then re-streaked on BBM plates till four generations to confirm the growth. These colonies were then grown in BBM for further analysis. 2.2 Growth and Dry cell weight (DCW) analysis Three fast growing mutants (CM2, CM5 and CM7) were able to survive the EMS treatment and therefore selected for growth/biochemical analysis. The mutants were cultivated in 500 mL Erlenmeyer flask containing 250 ml BBM for a period of 10 days under cool white fluorescent light (200 µmol m-2 s-1) at 27 °C. The growth rate was measured by taking the absorbance at 750 nm every 24 h and compared with the wild type. The dry cell weight (DCW; g/L) was measured on the 10th day by centrifuging the mutants and the wild type cells at 8000 rpm for 10 min. The wet biomass was dried at 80°C for 2 h in hot air oven and then gravimetrically weighed to get the DCW. Biomass productivity was then calculated using the following equation: Biomass Productivity = DCW/ cultivation time (days)
2.3 Starch and Lipid analysis
6
To estimate the starch content in the mutants as compared to the wild type, iodine staining was performed on the 10th day. Briefly, 500 µL of cell culture was mixed with 10 µL of iodine solution (10 mL distilled water, 0.5 g Iodine and 1g KI at 90 °C) and then centrifuged at 8000 rpm for 10 min (Takeshita et al., 2015). The cell pellet was washed thrice with 0.1% NaCl before visualizing under light microscope (EVOS-FL, Advance Microscopy Group, AMG, USA). Nile red staining was done to visualize lipid droplets inside the microalgal cells (Arora et al., 2016a). The total lipid was extracted using modified protocol of Bligh and Dyer (Bligh and Dyer, 1959; Arora et al., 2016a). Lipid productivity was then calculated according to the following equation: Lipid content (%) = Total lipid in grams / DCW Lipid Productivity = Lipid content (%) * Biomass Productivity/100 To detect the presence of triacylglycerols (TAGs) in the total lipids, thin layer chromatography (TLC) and Fourier Transform Infrared (FTIR) spectroscopy were performed. For TLC, 5 µl of the total lipid sample was spotted on the TLC plate (0.25-mm-thick silica gel G-60, F254 TLC plate; Merck, India) as described by Patel et al., 2015. FTIR spectra (Thermo Nicolet NEXUS, Maryland, USA) were collected over the wave number range 400–4000 cm-1 and triolein was used as standard (Miglio et al., 2013). 2.4 Biochemical and Morphological analysis In order to visualize the morphological changes in the mutants of C. minutissima as compared to its wild type, FESEM analysis was performed using previously described protocol (Arora et al., 2016b). The cell size (average of 100 cells) was also measured using ‘Image J 1.49a’ software and compared with the wild type (Patel et al., 2015).
7
The photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) were estimated on the 10th day by harvesting 2 ml of cell suspension, then adding 2 ml methanol (99.9 %) to the pellet and incubating it at 45 °C for 24 h. The supernatant was used to calculate the pigments as using the following equations: Chlorophyll a (Chl a; µg/ml) = 16.72 A665.2 – 9.16 A652.4 Chlorophyll b (Chl b; µg/ml) = 34.09 A652.4 – 15.28 A665.2 Carotenoids (µg/ml) = (1000 A470 –1.63 Chl a –104.9 Chl b)/221 where A is the absorbance (Lichtenthaler, 1987) Total carbohydrate content in the mutants and wild type microalgal cells were estimated using phenol sulfuric method (Dubois et al., 2002). CHNS elemental analyzer (Thermo-Scientific, USA) was used to estimate the total nitrogen content and then multiplied by a factor of 6.25 for total protein estimation. 2.5 Fatty acid methyl ester (FAME) analysis The total lipid extracted was transesterified using 6 % methanolic H2SO4 at 90 °C for 2 h (Arora et al., 2016a). The FAME phase was separated by mixing in hexane: water (1:2; v/v) followed by centrifugation at 8000 rpm for 5 min. The hexane containing FAME phase was expatriated out into fresh glass vial and 1µl of the FAME was used for Gas chromatography- mass spectroscopy analysis (GC-MS; Agilent Technologies, USA) with electron ionization (70 eV), DB-5 capillary column (30 mm *0.25 mm* 1 µm) using helium (1 ml/min) as carrier gas (Arora et al., 2016b). The biodiesel physical properties such as saponification value (mg KOH), Iodine value (g I2/100g), cetane number, degree of unsaturation (% wt), long chain saturation factor (% wt), high heating value (MJ/kg), kinematic viscosity (mm2 /s), density(g/cm3) and oxidative stability (h) 8
were estimated according to the empirical formulas and compared to plant oil methyl esters (Jatropha and Palm) as described previously (Arora et al., 2016a). Results and Discussion 3.1 Survival rate exhibited by EMS mutants Mutagenesis is a simple and robust technique to generate mutants of desirable characteristics such as high growth, lipid, carotenoid, thermo tolerance, heavy metal tolerance etc. (GarcíaVillada et al., 2004; Kamath et al., 2008; Ong et al., 2010; Ngampuak et al., 2016; Patel et al., 2016; Sirikhachornkit et al., 2016). High lipid accumulating microalgal strain development can lead to successful deployment of microalgal oil derived biodiesel on a large scale. EMS is a strong chemical mutagen as compared to UV radiations and has been widely used to generate microalgal mutants (Tillich et al., 2012). Keeping this in view, random mutants of C. minutissima were generated using EMS. The microalgal cells were treated with different concentrations of EMS (0.4 -2.4 M) to analyze the cell survival and optimize EMS concentration for 5 % survival rate (Supplementary Fig. 1). An apparent decline in the cells was observed when EMS concentration was periodically increased with 2.4 M showing 100 % cell lethality. EMS (2M) showed 5% survival rate which was then used as benchmark for further mutant generations. Amid the colonies that survived the 2 M dose of mutagenic agent, seven fast growing (CM1, CM2, CM3, CM4, CM5, CM6, CM7) colonies were selected and re-streaked to confirm the fast growth and mutation. Out of these seven fast growing mutants, only three colonies (CM2, CM5, and CM7) were able to survive on sub culturing (Supplementary Fig. 1). All these three rapid growing mutant strains were then evaluated for various growth/biochemical properties by cultivating them in 250 ml BBM separately.
9
3.2. Changes in the growth, dry cell weight and biomass productivity of the EMS mutants Based on the growth and appearance of microalgal colonies on BBM agar plates, all the three colonies (CM2, CM5 and CM7) were selected and compared to wild type for growth, DCW and productivity analysis. The growth rates of the mutants were recorded higher as compared to the wild type (Fig. 1). Among these mutants, CM7 showed the maximum absorbance at 750 nm followed by CM5 and CM2 as depicted in Fig. 1. The high growth rate is directly correlated with enhanced DCW and biomass productivity obtained by CM7 (2.4 ±0.2 g/L; 240 mg/L/d) followed by CM5 (2.2 ±0.25 g/L; 220 mg/L/d) and CM2 (1.67 ±0.2 g/L; 167 mg/L/d) respectively as compared to control (wild type species; 1.5 ±0.1 g/L; 150
mg/L/d) indicating that EMS
treatment has positively affected the growth metabolism (Table 1). The sharp increase in the growth rate of mutants may also indicate augmented metabolic rate in CM7. Indeed, similar sort of growth rate behavior has been reported for other green microalgae mutants such as Chlamydmonas reinhardtii, Synechocystis PCC 6803, C. pyrenoidosa NCIM 2738, Nannochloropsis sp.,
N. salina CCAP849/3 (Anandarajah et al., 2012; Lee et al., 2014;
Beacham et al., 2015; Patel et al., 2016; Sachdeva et al., 2016). In comparison to previous studies on EMS mutants of Nannochloropsis sp., Synechocystis PCC 6803 and Desmodesmus sp., CM7 showed higher biomass production (Table 1) (Doan and Obbard 2012; Patel et al., 2016; Zhang et al., 2016).
3.3 Changes in the starch and lipid composition in the EMS mutants The effects of EMS (2M) on starch and lipid content of CM2, CM5 and CM7 were observed by Iodine and Nile red staining. The mutants (CM5 and CM7) showed decreased starch granules 10
(blue spots) as compared to CM2 and control (Supplementary Fig. 2). Starch is a major form of carbohydrate storage and its synthesis directly competes with the lipid accumulation in microalgae as both pathways share common precursors (Sirikhachornkit et al., 2016; Patel et al., 2016). Their studies have shown that a decrease in the starch content or generation of starch less mutants led to increase in the lipid accumulation. Analogous to these studies, an increase in the intracellular lipid content in CM7 and CM5 was observed showing accumulation of lipid droplets (Supplementary Fig. 2). These qualitative results were in coherence with the total extracted lipid with CM7 showing ~1.5 folds enhanced lipid content (42 ± 0.25 %) and ~2.5 folds increase in the lipid productivity (101 ± 0.78 mg/L/d) followed by CM5 and CM2 as compared to the wild type (27 ± 0.2 % ; 41 mg/L/d) as shown in Table 1 and 2. A study on double mutants of Aradopsis thaliana suggested the possible role of ESV1 and LESV proteins involved in starch degradation (Feike et al., 2016). We envisage that EMS exposure resulted in activation of starch degrading enzymes, which in turn could result in generation of mutants with high lipid content. Furthermore, mutations at one or more locus in fatty acid biosynthesis genes could also delineate for higher lipid accumulation in mutants obtained. DGAT1 (Diacylglycerol acetyl transferase) enzyme plays a vital role in lipid synthesis as it catalyzes the conversion of diacylglycerol (DAG) to triacylglycerol (TAG) in the endoplasmic reticulum, and genetic change in such genes could hold a possible reason for increase in lipid content (Perry et al., 1999).
3.4 Biochemical and morphological variation in the EMS mutants To understand the alterations in the generated mutants at the biochemical level, the changes in the total carbohydrate, protein and photosynthetic pigments (chlorophyll a, chlorophyll b and
11
carotenoids) were estimated and compared with wild type (Table 2). Decrease in the starch content indicated that the level of carbohydrates in the mutants should be low as carbohydrate is majorly stored in the form of starch granules. Among the mutants, CM7 showed the lowest carbohydrate content (~1.7 folds lower) than wild type followed by CM5 and CM2 respectively (Table 2). The protein content in CM7 showed negligible change while CM2 and CM5 showed ~16-17 % decline as compared to wild type which deemed to be a possible explanation for increase in the growth rate of mutants (Table 2). Such dynamics of various biochemical properties could be the major reason that accounts for higher lipid accumulation in algal mutants. The mutants obtained on the plates showed considerable changes in the level of pigments present indicating the efficiency of photosynthesis receptors, which can be used to measure the physiological adaptivity. Chlorophyll content measures the physiological adaptivity of the mutants with the genetic variability supposed to be established due to the mutagenic agent (Fan et al., 2014). Interestingly, CM7 showed elevated levels of photosynthetic pigments such as chlorophyll a (~1.3 folds), chlorophyll b (~1.2 folds) as compared to wild type while similar carotenoids content were observed in them (Table 2). This was also evident from the color of the mutants which appeared more dark green as compared to wild type (Supplementary Fig 1). Similar results were obtained in EMS treated mutants of Synechocystis PCC 6803 which reported increase in the pigments of the mutants (Patel et al., 2016). This increase in biomass as well as chlorophyll content in CM7 could account for multifarious changes in the genome of the algae strain which may result in such phenotypic variations. Further, an increase in the level of chlorophyll a could result in the up regulation in the activity of ACCase (committing step for fatty acid synthesis) which could be a possible explanation for high lipid content (Lv et al., 2010). Presence of LHCII complex marks a remarkable effect on the photosynthetic ability of 12
microalgae (Drop et al., 2014). The data suggested that either due to modification in starch synthesis enzymes or polygenic point mutation in lipid generating pathway could be a possible reason for high lipid accumulating mutants. Such diverse phenotypic effects of EMS could also attribute for the polygenic mutation caused in C. minutissima. Microscopic imaging (light microscopy and FESEM) were done to analyze the alterations in the appearance and cell size of the mutants (Supplementary Fig. 3). Images visualized showed no change in the appearance of the mutants cells (CM2, CM5 and CM7) as compared to the wild type. It could be possible that EMS led to point mutation in the algal cells resulting in changes at the molecular level with no visible cell size and morphological changes (Table 2). 3.5 Analysis of triacylglycerol in the total lipid extracted and FAME profile of EMS mutant Transesterification of TAGs results in the formation of biodiesel. For high grade biodiesel quality, TAG content present in microalgae cell plays the key role (Krohn et al., 2011). Thus, the total lipid accumulated in mutant and wild type and the highest lipid content mutant CM7 were analyzed for TAG quality and properties using various biophysical techniques. Visible brown spot was observed on TLC plate which confirmed the presence of TAG in CM7 and wild type using Triolein as a control (Supplementary Fig. 4). Further, FTIR spectrum showed distinctive peaks at C-H bond vibrations mainly aliphatic chains of CH3 and CH2 at ~2925-2850 cm-1. Peak at ~1745 cm-1 , ~ 1160 cm-1 confirmed the presence of C=O and C-O-C stretch which correspond to the ester groups of TAGs. Absence of bands in the spectral region 4000–3450 cm-1 indicated lack of hydroxyl and amine groups (Supplementary Fig. 4). Results obtained here were in coherence with the study of Miglio et al. indicating presence of TAGs in the total lipid extracted (Miglio et al., 2013).
13
The FAME profile of transesterified samples from CM7 and wild type revealed the presence of myristic acid (C14:0), palmitic acid (C16:0), hexadecadienoic acid methyl ester (C16:1), heptadecanoic acid (C17:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and eicosanoic acid (C20:0) as shown in Fig. 2. The mutant strain (CM7) as compared to wild type showed decrease in saturated fatty acids (~1.1 folds), polyunsaturated fatty acids (~1.75 folds) while increase in mono-saturated fatty acids particularly C18:1 by ~1.6 folds respectively. This decreased level of poly unsaturated fatty acids (PUFA) can increase the storage life of the derived biodiesel from the mutant CM7, as high levels of the same (PUFA) could lead to oxidation resulting in the formation of epoxy fatty acids (Patel et al., 2016). Elevated content of mono unsaturated fatty acids (MUFA) recorded in CM7 could be responsible for ameliorating the use of biodiesel at low temperatures on account of its liquidity as has been reported by Zhang et al., 2016 while working on EMS mutants of Desmodesmus sp. Therefore, increased MUFA along with decreased PUFA content elucidate for better quality of biodiesel in CM7 as compared to wild type. The biodiesel physical properties obtained of the mutant (CM7) were better than wild type (WT) and abided by the standards given by ASTM D6751-02 and EN 14214 respectively (Table 3). CM7 mutant showed a low Iodine value (60 g I2/100g) with a high cetane number (67) and high heating value (41 MJ/kg) and oxidative stability (11 h) indicating shorter ignition delay time, better cold start behavior, smooth engine run, long shelf life and complete combustion leading to reduced gaseous and particulate emissions as compared to WT (Ramos et al., 2009; Arora et al., 2016a). Further, the density and kinematic viscosity of both WT and CM7 were within the permissible range (3.5 to 5.0 mm2/s; 0.86 to 0.90 g/cm3). The obtained biodiesel physical
14
properties were also comparable to plant oil methyl esters (Jatropha and Palm) as shown in Table 3. 4. Conclusion This study reported the generation of mutant strain (CM7) with higher growth rate and elevated level of lipid content of 42 ± 0.2 % with no significant changes in its morphology and size. The mutant exhibited reduced starch (carbohydrate content) and increased photosynthetic pigments that can be attributed to its high growth rate. However, to deduce the molecular level changes related to the enhancement of lipid product in the CM7 mutant strain, studies comprising of metabolomics and proteomic are quintessential. Further, in order to explore the mutant’s field applicability, environmental and ecological acceptability must be established. Acknowledgement Authors are thankful for financial support by the Department of Biotechnology, Govt. of India, BioCare Programme, DBT Sanction No. 102/IFD/SAN/3539/2011-2012 (Grant No. DBT-608BIO) to PAP and DBT-SRF to NA (Grant No. 7001-35-44). KMP acknowledges the receipt of DBT-IYBA fellowship and SERB-LS young scientist award. Figure Legends: Fig. 1: Growth curves of C. minutissima wild type (WT) and its mutants (CM2, CM5 and CM7) grown in BBM for 10 days. Fig 2: FAME profile of the extracted lipid from the wild type and CM7.
References
15
1. Anandarajah, K., Mahendraperumal, G., Sommerfeld, M., Hu, Q., 2012. Characterization of microalga Nannochloropsis sp. mutants for improved production of biofuels. Appl. Energy 96, 371–377. doi:10.1016/j.apenergy.2012.02.057 2. Arora, N., Patel, A., Pruthi, P.A., Pruthi, V., 2016a. Synergistic dynamics of nitrogen and phosphorous influences lipid productivity in Chlorella minutissima for biodiesel production. Bioresour. Technol. 213, 79-87. 3. Arora, N., Patel, A., Pruthi, P.A., Pruthi, V., 2016b. Boosting TAG accumulation with improved biodiesel production from novel oleaginous microalgae Scenedesmus sp. IITRIND2 utilizing waste sugarcane bagasse aqueous extract (SBAE). Appl Biochem Biotechnol. 180,422 109-121. 4. Beacham, T.A., Macia, V.M., Rooks, P., White, D.A., Ali, S.T., 2015. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis. Biotechnol. Reports 7, 87–94. doi:10.1016/j.btre.2015.05.007 5. Bligh, E., Dyer, W., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. 37. 6. Doan, T.T.Y., Obbard, J.P., 2012. Enhanced intracellular lipid in Nannochloropsis sp. via random mutagenesis and flow cytometric cell sorting. Algal Res. 1, 17–21. doi:10.1016/j.algal.2012.03.001 7. Drop, B., Webber-Birungi, M., Yadav, S.K.N., Filipowicz-Szymanska, A., Fusetti, F., Boekema, E.J., Croce, R., 2014. Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii. Biochim. Biophys. Acta Bioenerg. 1837, 63–72. doi:10.1016/j.bbabio.2013.07.012 8. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 2002. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. doi:10.1021/ac60111a017 9. Fan, J., Cui, Y., Wan, M., Wang, W., Li, Y., 2014. Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors. Biotechnol Biofuels 7, 17. doi:10.1186/1754-6834-7-17 10. Feike, D., Seung, D., Graf, A., Bischof, S., Ellick, T., Coiro, M., Soyk, S., Eicke, S., Mettler-Altmann, T., Lu, K.-J., Trick, M., Zeeman, S.C., Smith, A.M., 2016. The starch granule-associated protein EARLY STARVATION1 is required for the control of starch 16
degradation
in
Arabidopsis
thaliana
leaves.
Plant
Cell
28,
1472–1489.
doi:10.1105/tpc.16.00011 11. García-Villada, L., Rico, M., Altamirano, M., Sánchez-Martín, L., López-Rodas, V., Costas, E., 2004. Occurrence of copper resistant mutants in the toxic cyanobacteria Microcystis aeruginosa: Characterization and future implications in the use of copper sulphate as algaecide. Water Res. 38, 2207–2213. doi:10.1016/j.watres.2004.01.036 12. Krohn, B.J., McNeff, C. V., Yan, B., Nowlan, D., 2011. Production of algae-based biodiesel using the continuous catalytic Mcgyan process. Bioresour. Technol. 102, 94– 100. doi:10.1016/j.biortech.2010.05.035 13. Lam, M.K., Lee, K.T., 2012. Microalgae biofuels: A critical review of issues, problems and
the
way
forward.
Biotechnol.
Adv.
30,
673–690.
doi:10.1016/j.biotechadv.2011.11.008 14. Lee, B., Choi, G.G., Choi, Y.E., Sung, M., Park, M.S., Yang, J.W., 2014. Enhancement of lipid productivity by ethyl methane sulfonate-mediated random mutagenesis and proteomic analysis in Chlamydomonas reinhardtii. Korean J. Chem. Eng. 31, 1036–1042. doi:10.1007/s11814-014-0007-5 15. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. doi:10.1016/0076-6879(87)48036-1 16. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. doi:10.1016/j.biortech.2010.03.120 17. Ma, Y., Wang, Z., Zhu, M., Yu, C., Cao, Y., Zhang, D., Zhou, G., 2013. Increased lipid productivity and TAG content in Nannochloropsis by heavy-ion irradiation mutagenesis. Bioresour. Technol. 136, 360–367. doi:10.1016/j.biortech.2013.03.020 18. Miglio, R., Palmery, S., Salvalaggio, M., Carnelli, L., Capuano, F., Borrelli, R., 2013. Microalgae triacylglycerols content by FT-IR spectroscopy. J. Appl. Phycol. 25, 1621– 1631. doi:10.1007/s10811-013-0007-6 19. Ngampuak, V., Chookaew, Y., Dejtisakdi, W., 2016. KU11 Mutants affected for growth rate , cell accumulation and biomass. WASET. 10, 420–424. 20. Ong, S.C., Kao, C.Y., Chiu, S.Y., Tsai, M.T., Lin, C.S., 2010. Characterization of the thermal-tolerant mutants of Chlorella sp. with high growth rate and application in 17
outdoor
photobioreactor
cultivation.
Bioresour.
Technol.
101,
2880–2883.
doi:10.1016/j.biortech.2009.10.007 21. Patel, A., Sindhu, D.K., Arora, N., Singh, R.P., Pruthi, V., Pruthi, P.A., 2015. Biodiesel production from non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp using oleaginous yeast Rhodosporidium kratochvilovae HIMPA1. Bioresour. Technol. 197, 91– 98. doi:10.1016/j.biortech.2015.08.039 22. Patel, V.K., Maji, D., Pandey, S.S., Rout, P.K., Sundaram, S., Kalra, A., 2016. Rapid budding EMS mutants of Synechocystis PCC 6803 producing carbohydrate or lipid enriched biomass. Algal Res. 16, 36–45. doi:10.1016/j.algal.2016.02.029 23. Perry, H.J., Bligny, R., Gout, E., Harwood, J.L., 1999. Changes in Kennedy pathway intermediates associated with increased triacylglycerol synthesis in oil-seed rape. Phytochemistry 52, 799–804. doi:10.1016/S0031-9422(99)00294-0 24. Radakovits, R., Jinkerson, R.E., Fuerstenberg, S.I., Tae, H., Settlage, R.E., Boore, J.L., Posewitz, M.C., 2012. Draft genome sequence and genetic transformation of the oleaginous
alga
Nannochloropis
gaditana.
Nat.
Commun.
3,
686.
doi:10.1038/ncomms1688 25. Ramos, M.J, Fernández, C.M., Casas, Rodríguez L., Pérez, Á., 2009. Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour. Technol, 100, 261– 268. doi.org/10.1016/j.biortech.2008.06.039 26. Sachdeva, N., Gupta, R.P., Mathur, A.S., Tuli, D.K., 2016. Enhanced lipid production in thermo-tolerant mutants of Chlorella pyrenoidosa NCIM 2738. Bioresour. Technol. 221, 576–587. doi:10.1016/j.biortech.2016.09.049 27. Sandesh Kamath, B., Vidhyavathi, R., Sarada, R., Ravishankar, G.A., 2008. Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus
pluvialis
mutants.
Bioresour.
Technol.
99,
8667–8673.
doi:10.1016/j.biortech.2008.04.013 28. Sharma, P., Jha, A.B., Dubey, R.S., Pessarakli, M., 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot. 2012, 1–26. doi:10.1155/2012/217037 29. Singh, A., Olsen, S.I., Nigam, P.S., 2011. A viable technology to generate thirdgeneration biofuel. J. Chem. Technol. Biotechnol. 86, 1349–1353. doi:10.1002/jctb.2666 18
30. Sirikhachornkit, A., Vuttipongchaikij, S., Suttangkakul, A., Yokthongwattana, K., Juntawong, P., Pokethitiyook, P., Kangvansaichol, K., Meetam, M., 2016. Increasing the triacylglycerol content in Dunaliella tertiolecta through isolation of starch-deficient mutants. J. Microbiol. Biotechnol. 26, 854–866. doi:10.4014/jmb.1510.10022 31. Takeshita, T., Takeda, K., Ota, S., Yamazaki, T., Kawano, S., 2015. A simple method for measuring the starch and lipid contents in the cell of microalgae. Cytologia (Tokyo). 80, 475–481. doi:10.1508/cytologia.80.475 32. Tang, H., Chen, M., Garcia, M.E.D., Abunasser, N., Ng, K.Y.S., Salley, S.O., 2011. Culture of microalgae Chlorella minutissima for biodiesel feedstock production. Biotechnol. Bioeng. 108, 2280–2287. doi:10.1002/bit.23160 33. Tillich, U.M., Lehmann, S., Schulze, K., Dühring, U., Frohme, M., 2012. The Optimal Mutagen Dosage to Induce Point-Mutations in Synechocystis sp. PCC6803 and Its Application
to
Promote
Temperature
Tolerance.
PLoS
One
7,
1–8.
doi:10.1371/journal.pone.0049467 34. Vonlanthen, S., Dauvillée, D., Purton, S., 2015. Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids. Algal Res. 12, 109– 118. doi:10.1016/j.algal.2015.08.008 35. Zhang, Y., He, M., Zou, S., Fei, C., Yan, Y., Zheng, S., Rajper, A.A., Wang, C., 2016. Breeding of high biomass and lipid producing Desmodesmus sp. by Ethylmethane sulfonate-induced
mutation.
Bioresour.
doi:10.1016/j.biortech.2016.01.120
19
Technol.
207,
268–275.
Fig. 1
20
21
Table 1: Comparison of previously reported algal EMS mutants with the current study
Microalgae
WT/ Mutant
Biomass (g/L)
Lipid (%)
Nannochloropsis sp.
WT Mutant WT Mutant WT Mutant WT Mutant WT Mutant WT Mutant (CM2) Mutant (CM5) Mutant (CM7)
0.20 ± 0.02 0.22 ± 0.08 2.7 3.1 2.9 ± 0.002 2.46 ± 0.006 0.534 ± 0.014 0.778 ± 0.012 1.182 ± 0.010 1.397 ± 0.011 1.5±0.1 1.67±0.2
Chlorella pyrenoidosa Desmodesmus sp. Synechocystis PCC 6803 C. minutissima
34 ± 3.8 50.8 ± 6.8 47.4 ± 0.02 48.3 ± 0.02 21.78 ± 0.023 39.89 ± 0.004 40.41 48.41 12.97 32.80 27±0.5 32±0.25
Biomass Productivity (mg/L/d) 11.12 12.22 456 ± 0.01 517 ± 0.03 580 492 28.15 ± 0.75 40.95 ± 0.64 84.43 99.78 150±1.2 167±2.3
Lipid Productivity (mg/L/d) 3.77 6.20 213 249.55 105.27 196.26 495 778.10 10.95 32.72 40.5±1.4 53.44±2.5
2.2±0.25
36±0.3
220±1.3
79.2±2.4
2.4±0.2
42±0.12
240±2.1
100.8±1.5
22
R
Table 2: Relative changes in cell size (µm), total biochemical composition (lipid, carbohydrate and protein) and photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) in C. minutissima and its mutants (CM2, CM5 and CM7). WT/Mutant
Wild type CM2 CM5 CM7
Cell size (µm) 5.1±0.1 4.8±0.3 4.9±0.2 5.0±0.3
Biochemical composition Total lipid (%) 27± 0.5 32±0.3 36±0.3 42±0.1
Total carbohydrate (%) 30.5±1.3 29.2±0.4 28.9±0.3 17.5±0.3
Photosynthetic pigments Total protein (%) 42.5±2.1 38.8±1.3 35.1±1.5 40.5±2.3
Chlorophyll a (µg/ml) 6.3±0.1 6.4±0.2 5.4±0.1 8.1±0.2
Chlorophyll b (µg/ml) 4.2±0.2 4.3±0.1 3.7±0.2 5.1±0.1
Carotenoids (µg/ml) 0.34±0.01 0.43±0.02 0.31±0.01 0.36±0.02
Table 3: Summary of the biodiesel physical properties of C. minutissima (Wild type and Mutant CM7) obtained in the current study and compared to ASTM D6751-52, EN 14214 biodiesel standards and plant oil methyl esters. Standard fuel parameters Physical properties Saponification value (mg KOH) Iodine value (g I2/100g) Cetane number Degree of unsaturation (% wt) Long chain saturation factor (% wt) High heating value (MJ/kg) Kinematic viscosity (mm2/s) Density(g/cm3 ) Oxidative stability (h)
ASTM D6751-02 47 min 1.9 to 6.0 -
C. minutissima
EN 14214 120 (max) 51 (min) 3.5 to 5.0 0.86 to 0.90 ≥6
23
Wild type 184±2 70±4 58±3 82±2 12±2 40±1 4.4±0.2 0.87±0.01 7±0.2
CM7 (Mutant) 184±3 60±2 67±1 75±2 10±0.4 41±0.5 4.3±0.1 0.87±0.02 11±0.1
Plant oil methyl esters JME 96 57 4.3 0.88 4
PME 49 42 4.5 0.92 16
-No standard limit designated by ASTM D6751-02 and EN 14214 biodiesel standards, JME= Jatropha methyl ester, PME= Palm oil methyl ester **As the values were calculated theoretically, the values were rounded off to whole numbers
24
Highlights • • • • •
High lipid accumulating mutants of C. minutissima using ethyl methyl sulphonate. Mutants exhibited enhanced growth rate and photosynthetic pigments Reduced starch content indicated re-allocation of carbon flux to lipids. Increased MUFA and decreased SFA/ PUFA content in mutant CM7. High MUFA improves shelf life, cold flow properties and cetane number of biodiesel.
25
Graphical Abstract
26